The present invention relates to novel 3-nitropyridine derivatives and the pharmaceutical compositions containing said derivatives. More specifically, the present invention relates to 3-nitropyridine derivatives and their pharmaceutically acceptable salts, represented by the following formula 1, which effectively inhibit proliferation of hepatitis B virus and human immunodeficiency virus. This invention also relates to the process for preparing 3-nitropyridine derivatives and to the pharmaceutical compositions containing said derivatives as effective ingredients against viruses. 
Wherein,
R1 is methoxy or 
R3 is H, hydroxy, dialkylamino group with C2xcx9cC6, straight or branched hydroxyalkyl group with C2xcx9cC6, straight or branched dihydroxyalkyl group with C3xcx9cC6, alkoxyalkyl group with C3xcx9cC6, or saturated or unsaturated 5or 6 membered heterocyclic compounds containing 1 to 3 heteroatoms selected from N, O, and S, which may be unsubstituted or substituted with alkyl group of C1xcx9cC3; R3 may or may not contain asymmetrical carbons;
R4 is H, straight or branched alkyl group with C1xcx9cC4, or cycloalkyl group with C3xcx9cC6;
R3 and R4 both may consist of 5 or 6 membered heterocyclic ring containing 1xcx9c3 heteroatoms selected from N, O, and S, which is unsubstituted or substituted with straight or branched alkyl group with C1xcx9cC5, straight or branched hydroxyalkyl group with C2xcx9cC5, or hydroxy;
R2 is indazol-5-yl, or indazol-6-yl;
n is an integer between 0 and 3.
Hepatitis B virus (HBV; referred as xe2x80x9cHBVxe2x80x9d hereinafter) causes acute or chronic hepatitis, which may progress to liver cirrhosis and liver cancer. It is estimated that three hundred million people are infected with HBV in the world (Tiollais and Buendia, Sci. Am., 264, 48, 1991). There has been much research about the molecular biological characteristics of HBV and their relationship to liver diseases in order to find ways to prevent and treat hepatitis B. Various vaccines and diagnostic drugs have been developed and much effort is being channeled into research to find treatment for hepatitis B.
HBV genome consists of genes for polymerase (P), surface protein (pre-S1, pre-S2 and S), core protein (pre-C and C), and X protein. Of these proteins expressed from HBV genes, polymerase, surface protein, and core protein are structural proteins and X protein has a regulatory function.
The gene for HBV polymerase comprises 80% of the whole virus genome and produces a protein of 94 kD size with 845 amino acids, which has several functions in the replication of virus genome. This polypeptide includes sequences responsible for activities of protein primer, RNA dependent DNA polymerase, DNA dependent DNA polymerase, and RNase H. Kaplan and his coworkers first discovered reverse transcriptase activities of polymerase, which led to much research in replicating mechanism of HBV.
HBV enters liver when antigenic protein on virion surface is recognized by hepatic cell-specific receptor. Inside the liver cell, DNAs are synthesized by HBV polymerase action, attached to short chain to form complete double helix for HBV genome. Completed double helical DNA genome of HBV produces pre-genomic mRNA and mRNAs of core protein, surface protein, and regulatory protein by the action of RNA polymerase. Using these mRNAs, virus proteins are synthesized. Polymerase has an important function in the production of virus genome, forming a structure called replicasome with core protein and pre-genomic mRNA. This process is called encapsidation. Polymerase has repeated units of glutamic acid at the 3xe2x80x2-end with high affinity for nucleic acids, which is responsible for facile encapsidation. When replicasome is formed, (xe2x88x92) DNA strand is synthesized by reverse transcribing action of HBV polymerase and (+) DNA strand is made through the action of DNA dependent DNA polymerase, which in turn produces pre-genomic mRNAs. The whole process is repeated until the pool of more than 200 to 300 genomes is maintained (Tiollais and Buendia, Scientific American, 264: 48-54, 1991).
Although HBV and HIV are different viruses, the replication mechanisms during their proliferation have some common steps, namely, the reverse transcription of virus RNA to form DNA and the removal of RNA strand from subsequently formed RNA-DNA hybrid.
Recently, nucleoside compounds such as lamivudine and famvir have been reported to be useful inhibitors of HBV proliferation, although they have been originally developed as therapeutics for the treatment of acquired immune deficiency syndrome (AIDS; referred as xe2x80x9cAIDSxe2x80x9d hereinafter) and herpes zoster infection (Gerin, J. L, Hepatology, 14: 198-199, 1991; Lok, A. S. P., J. Viral Hepatitis, 1: 105-124, 1994; Dienstag, J. L. et al., New England Journal of Medicine, 333: 1657-1661, 1995). However, these nucleoside compounds are considered a poor choice for treatment of hepatitis B because of their high cost and side effects such as toxicity, development of resistant virus and recurrence of the disease after stopping treatment. Effort to find therapeutics for hepatitis B among non-nucleoside compounds has been continued and antiviral effects against HBV have been reported for quinolone compounds (EPO0563732, EPO0563734), iridos compounds (KR 94-1886), and terephthalic amide derivatives (KR 96-72384, KR 97-36589, KR 99-5100). In spite of much effort, however, effective drugs for treating hepatitis B have not been developed yet and therapeutic method mainly depends on symptomatic treatment.
AIDS is a disease inducing dramatic decrease in immune function in the body cells and causing various symptoms of infection rarely seen in normal human beings, which spread to the whole body. Human immunodeficiency virus (HIV; referred as xe2x80x9cHIVxe2x80x9d hereinafter) responsible for AIDS is known to mainly attack helper T cells, which is one of the T cells with regulatory function in the immune system. When helper T cells are infected with HIV virus and undergo necrosis, human immune system cannot function properly. Impairment in immune function subsequently results in fatal infection and development of malignant tumor. Since AIDS patient has been found in USA in 1981 for the first time, the number increased to more than 850,000 patients in 187 countries in 1993 (WHO 1993 report). WHO predicted that 30 to 40 million more people would be infected with HIV by the year 2000 and 10 to 20 million of them would develop the disease.
At the present time, drugs controlling proliferation of HIV have been most widely used for the treatment of AIDS. Of these, Zidovudine, which had been named Azidothymidine previously, is a drug developed in 1987. Didanosine was developed in 1991 as an alternative medicine for AIDS patients when Zidovudine was either ineffective or could not be used due to side effects. In addition, Zalcitabine was approved for concurrent use with Zidovudine in 1992. These drugs alleviate symptoms, slow down progression of the disease in the infected individuals to full-blown AIDS, and somewhat extend life span in the patients. These drugs, however, are not able to cure the patients completely and often develop problems such as resistance and side effects.
In light of these problems, we, inventors of the present invention, tried to develop therapeutics to treat hepatitis B with little chance of toxicity, side effects, and development of resistant viral strains. We found non-nucleoside compounds with excellent antiviral effect against HBV; synthesized novel 3-nitropyridine derivatives represented in formula 1 and completed the invention by showing their dramatic inhibitory effect on proliferation of HIV as well as of HBV.
The present invention provides novel 3-nitropyridine derivatives and the pharmaceutical compositions containing said derivatives. More specifically, the present invention provides 3-nitropyridine derivatives and their pharmaceutically acceptable salts, the process for their preparation and the pharmaceutical compositions containing said derivatives as effective ingredient. 3-nitropyridine derivatives of the present invention inhibit proliferation of hepatitis B virus as well as of human immunodeficiency virus and may be effectively used for prevention and treatment of hepatitis B and AIDS.
In order to accomplish the aforementioned goal, the present invention provides novel 3-nitropyridine derivatives represented below in formula 1 and their pharmaceutically acceptable salts. 
Wherein,
R1 is methoxy or 
R3 is H, hydroxy, dialkylamino group with C2xcx9cC6, straight or branched hydroxyalkyl group with C2xcx9cC6, straight or branched dihydroxyalkyl group with C3xcx9cC6, alkoxyalkyl group with C3xcx9cC6, or saturated or unsaturated 5 or 6 membered heterocyclic compounds containing 1 to 3 heteroatoms selected from N, O, and S, which may be unsubstituted or substituted with alkyl group with C1 xcx9cC3;
R3 may or may not have asymmetrical carbons;
R4 is H, straight or branched alkyl group with C1xcx9cC4, or cycloalkyl group with C3xcx9cC6;
R3 and R4 both may consist of 5 or 6 membered heterocyclic ring with 1 to 3 heteroatoms selected from N, O, and S, which is either unsubstituted or substituted with straight or branched alkyl group with C1xcx9cC5, straight or branched hydroxyalkyl group with C2xcx9cC5, or hydroxy;
R2 is indazol-5-yl, or indazol-6-yl;
n is an integer between 0 and 3.
When both R3 and R4 are represented as a 5 or 6 membered heterocyclic compounds with 1 to 3 heteroatoms selected from N, O, and S, n equals 0. This heterocyclic ring may be unsubstituted or substituted with straight or branched alkyl group with C1xcx9cC5, straight or branched hydroxyalkyl group with C2xcx9cC5, or hydroxy group;
When compounds of formula 1 have asymmetrical carbons, they may exist as either R or S optical isomer and the present invention covers both optical isomers and the racemic mixture as well.
Indazol-5-yl and indazol-6-yl groups for R2 in the present invention are represented in formula 2 and 3 respectively. 
Compounds of formula 1 in the present invention may be utilized in the form of salts and the acid addition salts prepared by adding pharmaceutically acceptable free acids are useful. Compounds of formula 1 may be changed to the corresponding acid addition salts according to the general practices in this field. Both inorganic and organic acids may be used as free acids in this case. Among inorganic acids, hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid may be used. Among organic acids, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid and aspartic acid may be used.
The present invention also provides the process for preparing 3-nitropyridine derivatives of formula 1 as represented in scheme 1. 
Wherein, X is Cl or OCH3; R2R3, R4 and n are as defined in formula 1.
The process of preparation in the present invention comprises the following steps:
(Step 1) Synthesis of 3-nitropyridine derivatives of formula 6 by reacting 2-chloro-3-nitropyridine derivatives of formula 4 with 5-aminoindazole or 6-aminoindazole of formula 5 in a proper solvent under a basic condition at an appropriate temperature;
(Step 2) Synthesis of 3-nitropyridine derivatives of formula 1 by reacting 3-nitropyridine derivatives of formula 6 prepared in step 1 with appropriate amine compounds of formula 7 in a proper solvent under a basic condition at an appropriate temperature.
When R1 in the compound of formula 1 is a methoxy group, step 1 completes the synthesis of desired compound (X=OCH3). In this case, the present invention includes the method of preparing 6-methoxy-3-nitropyridine derivatives of formula 6 by reacting 2-chloro-6-methoxy-3-nitropyridine of formula 4 with 5-aminoindazole or 6-aminoindazole of formula 5 in the presence of a base. 
Compound of formula 4 in the first step of scheme 1 is 2-chloro-6-methoxy-3-nitropyridine or 2,6-dichloro-3-nitropyridine.
Chemical reagents used in the first and the second steps of scheme 1, namely, 2-chloro-3-nitropyridine derivatives of formula 4, 5-aminoindazole or 6-aminoindazole of formula 5, and amine compounds of formula 7, are commercially available and may be purchased.
Compound of formula 7 in the step 2 above is used to introduce a substituent (R3xe2x80x94(CH2)nxe2x80x94NR4xe2x80x94) into the compound of formula 1 and an appropriate amine compound should be selected depending on the substituent desired, which can be easily done by one with general knowledge in the technical field.
To give more specific details about step 1 in the synthetic process, an organic base may be used and common tertiary amines such as triethylamine, N,N-diisopropylethylamine, N-methylmorpholine, N-methylpiperidine, 4-dimethylaminopyridine, N,N-dimethylaniline, 2,6-lutidine, pyridine are preferable.
Preferable reaction time and temperature are 4xcx9c15 hrs and 20xcx9c60xc2x0 C.
Preferable for the reaction is a single or a mixture of solvents selected from chloroform, methylene chloride, acetonitrile and alcohols such as methanol and ethanol.
Of 3-nitropyridine derivatives of formula 6 produced in the reaction of step 1, one with chloro group at 6 position is used in the following reaction of step 2.
The reaction in step 2 is described in more detail. Preferable solvent is a single or a mixture of solvents selected from acetonitrile, chloroform, methylene chloride, tetrahydrofuran, N,N-dimethylformamide, N-methylpyrrolidinone, pyridine, water and alcoholic solvents such as methanol, ethanol, and isopropanol.
It is preferable to use excess amount of amine compound (formula7) to increase the efficiency of the reaction. Solvents used in the previous step 1 for the synthesis of 3-nitropyridine derivatives of formula 6 are preferable. Reaction temperature of 25xcx9c80xc2x0 C. is preferable although it depends on the kind of amine compound used.
In another aspect of this invention, also provided are the pharmaceutical compositions of therapeutics for preventing and treating hepatitis B, which contain 3-nitropyridine derivatives of formula 1 and their pharmaceutically acceptable salts as effective ingredients.
The present invention also provides the pharmaceutical compositions of therapeutics for preventing and treating AIDS, which contain 3-nitropyridine derivatives and their pharmaceutically acceptable salts of formula 1 as effective ingredients.
3-nitropyridine derivatives of formula 1 in this invention have inhibitory effect on proliferation of both HIV and HBV because they interfere with removal of RNA strand from RNA-DNA hybrid formed during the reverse transcription of viral RNA to DNA, which is a common step in the replication mechanism of the two viruses.
Compounds of formula 1 may be taken orally as well as through other routes in clinical uses; for example, it may be administered intravenously, subcutaneously, intraperitoneally, or locally and used in the form of general drugs.
For clinical use of drugs with the pharmaceutical compositions of the present invention, compounds of formula 1 may be mixed with pharmaceutically acceptable excipients and made into various pharmaceutically acceptable forms; for example, tablets, capsules, trochese, solutions, suspensions for oral administration; and injection solutions, suspensions, or dried powder to be mixed with distilled water for the formulation of instant injection solution.
Effective dosage for compound of formula 1 is generally 10xcx9c500 mg/kg, preferably 50xcx9c300 mg/kg for adults, which may be divided into several doses, preferably into 1xcx9c6 doses per day if deemed appropriate by a doctor or a pharmacist.